The goals of the proposed research are to (1) to derive a set of suitable atomic radii for use in continuum dielectric Poisson-Boltzmann (PB) and solvent accessibility (SA) implicit solvent calculations within the context of a Drude polarizable force field and (2) apply this extended PB-polarizable force field treatment to RNA molecules of increasing complexity to quantify the driving forces for RNA folding, stability, and dynamics. The overarching objective is to study RNA folding by quantifying the free energy differences between conformational states and thus describe folding pathways for RNA in a quantitative manner. The PB/SA methodology could also be used in protein simulations. Simulations of RNA folding will be conducted using enhanced sampling methods to investigate folded, unfolded, and intermediate states of RNA molecules with various features (hairpins, pseudoknots, etc). Free energies from the MM/PBSA calculations will be coupled with information on base stacking energetics from quantum mechanics (QM) calculations to obtain a quantitative molecular understanding of events occurring during RNA folding. This information is important not only from a fundamental standpoint of understanding RNA folding, but also due to the fact that mutations in RNA that cause misfolding often lead to disease. In addition, studies on the SAM-II riboswitch, which binds S-adenosylmethionine (SAM) in bacteria, will be used to quantitatively describe the differences in apo- and SAM-bound configurations. Since many bacterial species use riboswitches to control gene expression, the proposed studies will provide information that can be used in the development of novel antibiotics. Polarizable force fields are especially relevant in these studies since the conformations of the strongly charged RNA molecules are highly dynamic and dependent upon metal binding. The three Aims described in this project are: 1. Extend the existing Drude polarizable force field to include parameters for MM/PBSA calculations. The use of MM/PBSA calculations allows for accurate estimates of free energies of macromolecular configurations. Atomic radii for MM/PBSA calculations will be tuned based on free energies of solvation from FEP and experiments. 2. Quantitate the effect of polarization on the folding and stabilization of small RNA molecules. RNA folding pathways are complex, and driving forces are not completely understood. Using enhanced sampling methods in conjunction with MM/PBSA and QM calculations, we will quantitate the role of polarization and metal binding on the folding pathway(s) of small RNA molecules. 3. Investigate the dynamics and free energy between conformational states of the SAM-II riboswitch. Riboswitch function depends on conformational changes induced by metabolite binding. In this Aim, we will investigate the driving forces behind these binding events and the resulting conformational changes.